Analyses of Molecular Mechanisms underlying Medulloblastoma Oncogenesis

虽然髓母细胞瘤(MB)的分子发病机制仍不清楚，但Wnt和Hedgehog发育信号通路的重要性最近已变得明显。为了进一步了解将正常小脑细胞转化为MB的分子事件，我们利用抑制消减杂交(SSH)技术来识别调控失调的分子，因此可能在MB的肿瘤发生中起重要作用。从相应的正常小脑组织中消减杂交后，分别构建了人和PTCH杂合子小鼠MBS的SSH文库。通过对文库的差异筛选，共分离到100多个在肿瘤组织中表达上调的基因片段，并进行了序列测定。使用NCBI BLAST程序对这些序列进行鉴定。我们选择了与肿瘤发生的重要过程有关的基因，如细胞增殖、细胞凋亡调节和小脑分化等，以进行进一步的分析。在人类MB文库中鉴定的基因包括Unc33-like蛋白(ULIP)、Sox4、Neuronatin、果蝇BarH-like 1的哺乳动物同源物(BARHL1)、核基质蛋白NRP/B(ENC1)和同源盒OTX2基因。在小鼠MB中发现上调的基因包括Cyclin D2、胸腺生成素、Musashi-1、蛋白磷酸酶2A抑制物2(I-2PP2A)和Unc5H4(D)。半定量RT-PCR结果显示，这些基因在人类MB中的表达水平明显高于正常小脑。此外，一些基因主要在MB中表达。这些基因的过度表达在正常细胞转化中所起的作用还有待充分确定。

Although the molecular pathogenesis of medulloblastoma (MB) remains poorly understood, the importance of the Wnt and Hedgehog developmental signaling pathways has recently become apparent. In order to further the understanding of molecular events that transform a normal cerebellar cell into a MB, we utilized the technique of suppression subtractive hybridization (SSH) to identify molecules that are dysregulated and therefore may be important in MB oncogenesis. After the subtractive hybridization of cDNA from corresponding normal cerebellar tissue, SSH libraries from both human and Ptch heterozygous murine MBs were generated. Through differential screening of the libraries, over 100 cDNA fragments up-regulated in the tumor were isolated and sequenced. These sequences were identified using the NCBI BLAST program. We selected genes involved in oncogenically important processes such as cellular proliferation, apoptosis regulation, and differentiation of the cerebellum to analyze further. Genes identified in the human MB library included Unc33-like protein (ULIP), SOX4, neuronatin, the mammalian homologue of Drosophila BarH-like 1(BARHL1), the nuclear matix protein NRP/B (ENC1), and the homeobox OTX2 gene. Genes found to be up-regulated in the murine MB included cyclin D2, thymopoietin, Musashi-1, protein phosphatase 2A inhibitor-2 (I-2PP2A), and Unc5H4 (D). Using semi-quantitative RT-PCR, the mRNA expression levels for these genes are markedly higher in human MB than in the normal cerebellum. Furthermore some genes were expressed predominantly in MB. The role played by the over-expression of these genes in the transformation of normal cells remains to be fully determined.